Luminescence-based Assays
Molecular luminescence is the emission of electromagnetic (EM) radiation, including ultraviolet (UV), visible and infrared (IR) light, from a molecule as it returns from an excited state to a lower energy state, usually the ground state of the molecule. Luminescence includes radioluminescence, chemiluminescence, which includes bioluminescence, and photoluminescence, which includes fluorescence and phosphorescence. Coupling of luminescent labels or reactions to assays, such as immuneassays, has provided convenient and sensitive assays. Chemiluminescence is produced when the excited product of an exoergic chemical process reverts to its ground state with the emission of light. Most chemiluminescent reactions require a step that involves oxidation of a reactant with molecular oxygen or its synthetic equivalent. Chemiluminescence is coupled to assays by using molecules, such as luminol, acridinium esters, isoluminol, lucigenin, dioxetanes and oxalate esters, that are capable of exhibiting chemiluminescence or transferring energy to an appropriate acceptor luminescent molecule or compound. The best known chemiluminescent reactions are those of the acridinium esters, of luminol and of lucigenin. In luminol, and isoluminol, chemiluminescent reactions, the key oxidative step involves reactions of hydrogen peroxide and aminophthalhydrazide in the presence of suitable catalysts.
Chemiluminescence is employed in several types of assays. It is employed in: (1) assays that use chemiluminescent labels, such as isoluminol derivatives or acridinium esters; (2) chemiluminescent assays that use agents, such as luminol, in combination with peroxide generating substrates, peroxidases and enzymes, such as oxidoreductases; (3) assays that involve electron transfer and fragmentation pathways that generate chemiluminescence; and (4) bioluminescence, which involves the use of biological substrates and enzymes, such as luciferin and luciferase, to generate light. Of particular interest herein, are chemiluminescent assays that are used to determine dehydrogenases and their cofactors NAD(P)/NAD(P)H.